1.Development and Optimization of Method for Generating Unmarked A. tumefaciens Mutants
Minliang GUO ; Qing ZHU ; Diankun GAO
Progress in Biochemistry and Biophysics 2009;36(5):556-565
Agrobacterium tumefaciens possesses many advantages as a model bacterium for the study of a wide variety of biological processes. Gene disruption or inactivation is a powerful and direct tool for investigation of in vivo gene functions. The intensive study ofA. tumefaciens has increased the need for simple and highly efficient procedures to manipulate its genome. The sacB gene was used as a counterselectable marker to develop a gene replacement procedure that allows precise insertion, deletion, and allele substitution of any gene sequence in A. tumefaciens without altering the genome in any other way. A kanamycin resistance (KmR) cassette was constructed to the suicide vector as the positive selection marker. The suicide plasmid containing DNA fragments homologous to the flanking sequences of the target gene was integrated into the recipient cell genome at the target gene locus by intermolecular homologous recombination, generating the KmR-single cross-over colonies. The effect of homologous sequence length on the intermolecular homologous recombination was analyzed. The second cross-over colonies generated by intramolecular homologous recombination occurring between two tandem repeats were simply screened out by counter-selection of sacB. Data showed that the intervening sequence length between two repeats significantly affected the intramolecular homologous recombination frequency in A. tumefaciens, indicating that A. tumefaciens adopted the homologous recombination mechanism similar to that in E. coli. All these results demonstrated that investigators could minimize the numbers of colonies to be analyzed and reduce the overall workload by optimizing the relative length of two homologous fragments and using the specific type of single cross-over transformants for screening the second cross-over event. This mutagenesis strategy had successfully been used to generate the double unmarked △vbp2△vbp3 mutant in two A. tumefaciens strains.
2.Quantitative evaluation of the left ventricular systolic dyssynchrony and its significance in patients with heart failure after myocardial infarction by real-time three-dimensional echocardiography
Qing DENG ; Qing ZHOU ; Limin ZHU ; Jinling CHEN ; Ruiqiang GUO
Chinese Journal of Ultrasonography 2010;19(8):662-665
Objective To quantitatively assess the left ventricular systolic dyssynchrony in patients with varied degrees of chronic congestive heart failure after old myocardial infarction(OMI) by real-time three-dimensional echocardiography(RT-3DE) and investigate the clinical value of the systolic dyssynchrony index(SDI). Methods Forty patients with congestive heart failure after OMI (infarction group) were divided into the severe dysfunction group (LVEF ≤35 %) and the mild dysfunction group (35 % < LVEF<50%) ,and 30 normal subjects served as the control. RT-3DE was performed on all subjects to obtain the 17-segmental time-volumetric curves and global systolic function. SDI changes in above groups and the correlation between SDI and LVEF were analyzed. Results The SDI of the infarction group was significantly higher than that of the normal control group ( P <0. 01 ). The SDI of the severe dysfunction group was significantly higher than that of the mild group (P<0.01). SDI and LVEF were negatively correlated ( r = -0.84, P <0. 01 ). The dyssynchrony rate in the infarction group was 85 %,in the severe dysfunction group was 100%, in the mild group was 75%. Conclusions Left ventricular systolic dyssynchrony is prevalent in patients with OMI, and it is negatively correlated with the LVEF. SDI is a sensitive indicator in assessing left ventricular systolic dyssynchrony. RT-3DE has a unique advantage in the evaluation of the left ventricular systolic dyssynchrony,especially in the patients with myocardial infarction.
3.Effect of hypoxia-inducible factor-1t expression in toll-like receptor 4 signaling pathway-mediated rat lung ischemia-reperfusion injury and possible mechanism
Zhiyi ZHOU ; Xingfeng ZHU ; Jingyu CHEN ; Qing GUO ; Guoyi YANG
Chinese Journal of Organ Transplantation 2014;35(9):561-566
Objective To investigate the effect of hypoxia-inducible factor-1α expression (HIF-1α) on toll-like receptor 4 (TLR4) signaling pathway-mediated rat lung ischemia-reperfusion injury (LIRI).Method Forty-five S-D rats were randomly divided into Sham group,LIRI group,LIRI+ TLR4-activated group,LIRI+ TLR4-inhibited group,LIRI + ASK1-inhibited group,LIRI + p38-inhibited group,and LIRI + HIF-1α-inhibited group.The interaction between TLR4 signaling pathway [including TLR4,myeloid differentiation factor 88 (MyD88),TIR-domain-containing adapter-inducing interferon-βTIR-domain-containing adapter-inducing interferon-β (TRIF),Apoptosis signal-regulating kinase 1 (ASK1) and p38] and HIF-1α and the role of TLR4-dependent HIF-1α in LIRI in vivo were analyzed.Result In LIRI,HIF-1α accumulation was induced in a TLR4-dependent fashion,and MyD88,but not TRIF,and activation of ASK1 and P38 were found to be critical for TLR4-mediated HIF-1α accumulation.HIF-1α protein played a critical role in TLR4-mediated lung injury of LIRI.HIF-1α up-regulated TLR4 expression in LIRI in a positive feedback manner.Conclusion We identify that HIF-1α has a damaging effect on TLR4 signaling pathway-mediated LIRI and TLR4-HIF-1 may synergistically involved in the development of LIRI.Therefore we suggest that the interaction between them may represent a novel therapeutic target for the development of novel target-based therapies of LIRI.
4.Preoperative Evaluation of Pancreatic Carcinoma with Multidetector Spiral CT
Xiaodong WANG ; Qing MA ; Yulin GUO ; Li ZHU
Journal of Practical Radiology 2001;0(08):-
Objective To study the value of MDCT in the preoperative assessment of resectability in patients with carcinoma of pancreas.Methods 53 cases of pancreatic carcinomas with intact MDCT materials proved operation and pathology were collected.Combining the transverse images and multiplanar reformation(MPR),maximum intensity projection(MIP),curved planar reconstruction(CPR) and volume rendering(VR),the resectable possibility of tumors were evaluated preoperatively,inclunding the relationship between tumors,vessels and organs,as well as metastases.Results Of 53 cases,16 had underwent radical resection,2 cases believed unresectable preoperatively,which were overestimating the degrees of vessels involved by tumors,37 cases had palliative operation,in which 5 cases were believed resectable preoperatively.Lymph node or liver metastases were missed in 2 cases and 3 cases were underestimating the degrees of vessels involved by tumor.Conclusion In evaluating the resectability of pancreatic carcinoma preoperatively,MDCT plays an important role.
5.Metabolic Flux Analysis of L-Arginine Fermentation in Corynebacterium glutamicum
Jin-Wei ZHU ; Qing-Shan CHEN ; Wei-Guo ZHANG ;
Microbiology 1992;0(03):-
In this paper, metabolic networks of the Corynebacterium glutamicum GWY020 and the two de-rivatives carrying additional mutations HUI821and GUI089 were established and modified. The concentra-tions of extra-cellular metabolites were determined under sub-steady-state (50 h~52 h) of the batch culture. The metabolic flux distribution maps of the three strains were obtained, compared and analyzed. These re-sults indicate that the introduction of analog supersensitive marker or analog resistant marker skew the metabolic flux towards the formation of L-Arginine. This study revealed the usefulness of the metabolic flux analysis as a tool for verification of existing production strains. The analysis may play an important role in helping us to rationally re-design metabolism for further improvement of fermentation process.
6.Proximal epithelioid sarcoma: a case report.
Qing-zhu WEI ; Yan-hui LIU ; Heng-guo ZHUANG ; Dong-lan LUO ; Xin-lan LUO
Chinese Journal of Pathology 2006;35(10):638-639
Antigens, CD34
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metabolism
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Cytokines
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metabolism
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Diagnosis, Differential
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Humans
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Immunohistochemistry
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Male
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Middle Aged
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Mucin-1
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metabolism
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Perineum
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pathology
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Phosphopyruvate Hydratase
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metabolism
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Sarcoma
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metabolism
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pathology
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surgery
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Vimentin
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metabolism
7.Genotypes of β-lactamases produced by Klebsiella pneumoniae
Yuanyu GUO ; Zeqing WEI ; Peiqiong ZHU ; Xiaoxing DU ; Qing YANG ; Ping SHEN ; Yunsong YU
Chinese Journal of Clinical Infectious Diseases 2010;3(3):138-141
Objective To investigate the genotypes of β-lactamases produced by Klebsiella pneumoniae.Methods Plasmid conjugation,PCR amplification,gene cloning and DNA sequencing,isoelectric focusing electrophoresis and extended-spectrum β-lactamase(ESBLs)confirmatory test were carried out for analyzing the encoding gene of β-lactamases in clinical strains of Klebsiella pneumoniae collected from hospital wards.Results Totally 75 clinical strains of Klebsiella pneumoniae were collected,in which 48 strains were confirmed to produce genotype of β-laetamases(64.0%),including 39 ESBLs-producing Btraim(52.0%).Among 48 strains,17 isolates(35.4%)carried 2 types of ESBLs genes,7(14.6%)carried 3 types of ESBL8 genes,and 5(10.4%)carried 4 types of ESBLs genes.CTX-M was the most comon type(30/48,62.5%),followed by TEM(26/48,54.2%)and SHV(25/48,52.1%).Among 9 isolates with DHA-1 AmpC β-laetamase,8 produced AmpC β-lactamases and ESBLs.Class A carbapenemase KPC-2 was produced in 3 isolates.False negative rate of ESBLs confirmatory test was 23.1%(9/39).Condusion Genotypes of β-lactamases produced by Klebsiella pneumoniae are complicated,which results in multi-drug resistance in clinic.
8.The basic strategies and research advances in the studies on glycosyltransferases involved in ginsenoside biosynthesis.
Hui-Chao LIANG ; Qing-Hua WANG ; Ting GONG ; Guo-Hua DU ; Jin-Ling YANG ; Ping ZHU
Acta Pharmaceutica Sinica 2015;50(2):148-153
Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways
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Ginsenosides
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biosynthesis
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Glycosyltransferases
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metabolism
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Panax
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chemistry
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Plants, Medicinal
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chemistry
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Synthetic Biology
9.Establishment of quality standard for Corni Fructus based on the multiple bioactive constituents.
Sheng GUO ; Jin-ao DUAN ; Shao-qing ZHU ; Da-wei QIAN
China Journal of Chinese Materia Medica 2015;40(15):3017-3021
This study is to establish the methods for determination of iridoid glycosides and triterpenic acids in Corni Fructus and provide technical support for the quality control of Corni Fructus. Morroniside, loganin and sweroside were determined by HPLC-UV method with acetonitrile and 0.1% formic acid as the mobile phase, and the detective wavelength was set at 240 nm. Oleanolic acid and ursolic acid were determined by HPLC-ELSD method with methanol-0.5% ammonium acetate (87:13) as the mobile phase. The results showed that the linear ranges of morroniside, loganin and sweroside were 5.335-213.4 mg · L(-1) (r = 0.9999), 5.515-220.6 mg · L(-1) (r = 1.0000), 1.992-79.68 mg · L(-1) (r = 1.0000), respectively. The average recoveries of the above three iridoid glycosides were 98.49%-99.28% with RSDs of recoveries being less than 2%. The linear ranges of oleanolic acid and ursolic acid were 7.74-154.8 mg · L(-1) (r = 0.9964), 10.82-216.4 mg · L(-1) (r = 0.9996), respectively. The average recoveries of the above two triterpenic acids were 98.11%-99.27% with RSDs of recoveries being less than 3%. The method established in this research is simple, rapid and reliable, and can be used for quality control of Corni Fructus. Furthermore, the research provided experimental data for the improvement of present quality standard of Corni Fructus, which has important significance to guarantee its quality and clinical curative effect.
Cornus
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chemistry
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Drug Stability
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Oleanolic Acid
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analysis
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Quality Control
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Triterpenes
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analysis